Single-molecule real-time sequencing identifies massive full-length cDNAs and alternative-splicing events that facilitate comparative and functional genomics study in the hexaploid crop sweet potato.

BACKGROUND: Sweet potato (Ipomoea batatas (L.) Lam.) is one of the most important crops in many developing countries and provides a candidate source of bioenergy. However, neither a complete reference genome nor large-scale full-length cDNA sequences for this outcrossing hexaploid crop are available, which in turn impedes progress in research studies in I. batatas functional genomics and molecular breeding. METHODS: In this study, we sequenced full-length transcriptomes in I. batatas and its diploid ancestor I. trifida by single-molecule real-time sequencing and Illumina second-generation sequencing technologies. With the generated datasets, we conducted comprehensive intraspecific and interspecific sequence analyses and experimental characterization. RESULTS: A total of 53,861/51,184 high-quality long-read transcripts were obtained, which covered about 10,439/10,452 loci in the I. batatas/I. trifida genome. These datasets enabled us to predict open reading frames successfully in 96.83%/96.82% of transcripts and identify 34,963/33,637 full-length cDNA sequences, 1,401/1,457 transcription factors, 25,315/27,090 simple sequence repeats, 1,656/1,389 long non-coding RNAs, and 5,251/8,901 alternative splicing events. Approximately, 32.34%/38.54% of transcripts and 46.22%/51.18% multi-exon transcripts underwent alternative splicing in I. batatas/I. trifida. Moreover, we validated one alternative splicing event in each of 10 genes and identified tuberous-root-specific expressed isoforms from a starch-branching enzyme, an alpha-glucan phosphorylase, a neutral invertase, and several ABC transporters. Overall, the collection and analysis of large-scale long-read transcripts generated in this study will serve as a valuable resource for the I. batatas research community, which may accelerate the progress in its structural, functional, and comparative genomics studies.

A comparative metabolomics study of flavonoids in sweet potato with different flesh colors (Ipomoea batatas (L.) Lam).

To study the diversity and cultivar-specific of phytochemicals in sweet potato, Liquid Chromatography-Electrospray Ionization-Mass Spectrometry was used to analyze the metabolic profiles of five sweet potato cultivars exhibiting different flesh colors: purple, yellow/orange, and white. A total of 213 metabolites, including 29 flavonoids and 27 phenolic acids, were characterized. The flavonoid profiles of the five different cultivars were distinguished using PCA, the results suggested the flesh color accounted for the observed metabolic differences. In addition to anthocyanins, quinic acids and ferulic acids were the prominent phenolic acids, O-hexoside of quercetin, chrysoeriol were the prominent flavonoids in sweet potato tubers, and they were all higher in the OFSP and PFSP than WFSP. The main differential metabolic pathways between the OFSP, PFSP and the WFSP included those relating to phenylpropanoid and flavonoid biosynthesis. This study provides new insights into the differences in metabolite profiles among sweet potatoes with different flesh colors.

Identification and analysis of glutathione S-transferase gene family in sweet potato reveal divergent GST-mediated networks in aboveground and underground tissues in response to abiotic stresses.

BACKGROUND: Sweet potato, a hexaploid species lacking a reference genome, is one of the most important crops in many developing countries, where abiotic stresses are a primary cause of reduction of crop yield. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in oxidative stress tolerance and cellular detoxification. RESULTS: A total of 42 putative full-length GST genes were identified from two local transcriptome databases and validated by molecular cloning and Sanger sequencing. Sequence and intraspecific phylogenetic analyses revealed extensive differentiation in their coding sequences and divided them into eight subfamilies. Interspecific phylogenetic and comparative analyses indicated that most examined GST paralogs might originate and diverge before the speciation of sweet potato. Results from large-scale RNA-seq and quantitative real-time PCR experiments exhibited extensive variation in gene-expression profiles across different tissues and varieties, which implied strong evolutionary divergence in their gene-expression regulation. Moreover, we performed five manipulated stress experiments and uncovered highly divergent stress-response patterns of sweet potato GST genes in aboveground and underground tissues. CONCLUSIONS: Our study identified a large number of sweet potato GST genes, systematically investigated their evolutionary diversification, and provides new insights into the GST-mediated stress-response mechanisms in this worldwide crop.

A Single Nucleotide Deletion in Gibberellin20-oxidase1 Causes Alpine Dwarfism in Arabidopsis.

Alpine dwarfism is widely observed in alpine plant populations and often considered a high-altitude adaptation, yet its molecular basis and ecological relevance remain unclear. In this study, we used map-based cloning and field transplant experiments to investigate dwarfism in natural Arabidopsis (Arabidopsis thaliana) accessions collected from the Swiss Alps. A loss-of-function mutation due to a single nucleotide deletion in gibberellin20-oxidase1 (GA5) was identified as the cause of dwarfism in an alpine accession. The mutated allele, ga5-184, was found in two natural Arabidopsis populations collected from one geographic region at high altitude, but was different from all other reported ga5 null alleles, suggesting that this allele has evolved locally. In field transplant experiments, the dwarf accession with ga5-184 exhibited a fitness pattern consistent with adaptation to high altitude. Across a wider array of accessions from the Swiss Alps, plant height decreased with altitude of origin, but fitness patterns in the transplant experiments were variable and general altitudinal adaptation was not evident. In general, our study provides new insights into molecular basis and possible ecological roles of alpine dwarfism, and demonstrates the importance of the GA-signaling pathway for the generation of ecologically relevant variation in higher plants.

Herkogamy and its effects on mating patterns in Arabidopsis thaliana.

The evolution of mating systems, which exhibit an extraordinary diversity in flowering plants, is of central interest in plant biology. Herkogamy, the spatial separation of sexual organs within flowers, is a widespread floral mechanism that is thought to be an adaptive trait reducing self-pollination in hermaphroditic plants. In contrast with previous studies of herkogamy that focused on plants with relatively large floral displays, we here characterized herkogamy in Arabidopsis thaliana, a model plant with a strong selfing syndrome. Developmental features, reproductive consequences, and genetic architecture of herkogamy were exploited using naturally variable A. thaliana accessions, under both greenhouse and natural conditions. Our results demonstrate that the degree of herkogamy can strongly influence the mating patterns of A. thaliana: approach herkogamy can effectively promote outcrossing, no herkogamy is also capable of enhancing the opportunity for outcrossing, and reverse herkogamy facilitates efficient self-pollination. In addition, we found that the expression of herkogamy in A. thaliana was environment-dependent and regulated by multiple quantitative trait loci. This study reveals how minor modifications in floral morphology may cause dramatic changes in plant mating patterns, provides new insights into the function of herkogamy, and suggests the way for dissecting the genetic basis of this important character in a model plant.

Functional diversity of CYCLOIDEA-like TCP genes in the control of zygomorphic flower development in Lotus japonicus.

CYCLOIDEA (CYC)-like TCP genes play key roles in dorsoventral differentiation of zygomorphic flowers in Papilionoideae legumes. In this study, we analyzed the kew mutants whose flowers lost lateral identity, and investigated the diverse functions of three LjCYC genes during zygomorphic flower development in the model legume Lotus japonicus. We showed that kew1 and kew3 are allelic mutants of LjCYC3, a CYC-like TCP gene. Through transgenic experiments, it was shown that LjCYC1 possesses dorsal activity similar to LjCYC2, and that LjCYC3 alone is sufficient to confer lateral activity, and an epistatic effect between dorsal and lateral activities was identified. Sequence analysis revealed a striking alteration at the 3' end of the LjCYC3 open reading frame (ORF) in comparison with those of LjCYC1 and LjCYC2 ORFs. Furthermore, it was found that LjCYC proteins could interact with each other and possess different activities by means of a transcriptional activity assay. Our data demonstrate that the sequence variation and the subsequent alteration of protein property play important roles in the functional diversity of different LjCYC genes in controlling zygomorphic flower development in Lotus japonicus.

[Study of mtDNA 12S rRNA A1555G, GJB2, GJB3 gene mutation in Uighur and Han people with hereditary nonsyndromic hearing loss in Xinjiang].

OBJECTIVE: To study mtDNA, GJB2, GJB3 and determine gene mutation situs and frequency in Uighur and Han people with hereditary nonsyndromic hearing loss, and to compare the differences of gene mutation situs and frequency between Uighur and Han people. METHODS: Blood samples were obtained from 93 patients (43 Uygur and 50 Han) with hereditary non-syndromic hearing loss and 110 normal people (56 Uygur and 54 Han). Genomic DNA was extracted from isolated leukocytes, and amplified by polymerase chain reaction (PCR). PCR products of GJB3 were sequenced directly; while PCR products of mitochondrial DNA 12S rRNA A1555G point mutations were analyzed by PCR-Alw26I digestion, and positive ones were further sequenced. GJB2 genes of 83 patients (43 Uygur and 40 Han) with hereditary non-syndromic hearing loss and 98 normal people (46 Uygur and 52 Han) were directly sequenced. RESULTS: Among GJB3 genes of 93 patients, 2 cases of 33C-T, 2 cases of of 766G-A, 7 cases of 357C-T, and 4 cases of 798C-T were detected. Mitochondrial DNA 12SrRNA A1555G mutation was detected in 8 patients (2 Uygur and 6 Han). Nine kinds of base changes of GJB2 were detected: 109G-A, 233-235delC, 79G-A, 196G-A, 341A-G, 564G-A, 380G-A, 71G-A, and 35delG. In the control group, detected GJB3 mutations included 4 cases of 357C-T, 5 cases of 798C-T, and 2 cases of 93C-T; while 9 kinds of base changes of GJB2 were detected: 341A-G, 380G-A, 457G-A, 79-GA, 109G-A, 281A-G, 21G-T, 171G-T, and 368C-A. For mtDNA 12SrRNA A1555G, the difference between study group of and control group of Han people was statistically significant (P < 0.05). For GJB2 mutation 79G-A, the difference between study group and control group was statistically significant (P < 0.05) in both Uygur and Han people; while for GJB2 mutation 341A-G, the difference in study group between Uygur and Han people was statistically significant (P < 0.05). And for GJB3 mutation 798C-T, the difference was statistically significant both between study group and control group, and between Uygur and Han people (P < 0.05). CONCLUSIONS: In Xinjiang, mutation rate was high for mtDNA 12SRNA A1555G. while GJB3 gene mutations were not the main cause of the hereditary nonsyndromic hearing loss. There were certain ethnic and geographical characteristics of GJB2and GJB3 mutations.

[Analysis of mtDNA 12SrRNA A1555G mutations of Uigur patients with nonsyndromic hereditary hearing loss in Xinjiang].

OBJECTIVE: To explore the incidence of Uigur patients with nonsyndromic hereditary hearing loss in Xinjiang, and to provide the basis for preventing deafness caused by aminoglycoside antibiotics. METHOD: The medical history of 51 Uigur deaf patients as the study group was collected in Xinjiang. Fifty-three Uigur normal people were selected as the control group in Xinjiang. Blood samples were obtained from them with informed consents. Genomic DNA was extracted from isolated leukocytes. The mitochondrial DNA fragments were amplified by polymerase chain reaction. mtDNA 12SrRNA A1555G mutation was detected using A1w26I restriction endonuclease digestion, followed by direct sequencing to identify the A1555G mutation. RESULT: The mtDNA A1555G mutation was detected in 2 Uigur patients, and both of them had used aminoglycoside antibiotics. CONCLUSION: There is no statistically significant difference between patients and normal people in Xinjiang. The mtDNA A1555G mutation is related to aminoglycoside antibiotics-induced deafness, which can cause genetic stisceptibility to aminoglycoside antibiotics ototoxicity. The incidence of mtDNA A1555G is lower than the average level of the overall Chinese deaf population.

Genetic control of floral zygomorphy in pea (Pisum sativum L.).

Floral zygomorphy (flowers with bilateral symmetry) has multiple origins and typically manifests two kinds of asymmetries, dorsoventral (DV) and organ internal (IN) asymmetries in floral and organ planes, respectively, revealing the underlying key regulators in plant genomes that generate and superimpose various mechanisms to build up complexity and different floral forms during plant development. In this study, we investigate the loci affecting these asymmetries during the development of floral zygomorphy in pea (Pisum sativum L.). Two genes, LOBED STANDARD 1 (LST1) and KEELED WINGS (K), were cloned that encode TCP transcription factors and have divergent functions to constitute the DV asymmetry. A previously undescribed regulator, SYMMETRIC PETALS 1 (SYP1), has been isolated as controlling IN asymmetry. Genetic analysis demonstrates that DV and IN asymmetries could be controlled independently by the two kinds of regulators in pea, and their interactions help to specify the type of zygomorphy. Based on the genetic analysis in pea, we suggest that variation in both the functions and interactions of these regulators could give rise to the wide spectrum of floral symmetries among legume species and other flowering plants.

Control of petal shape and floral zygomorphy in Lotus japonicus.

Zygomorphic flowers, with bilateral (dorsoventral) symmetry, are considered to have evolved several times independently in flowering plants. In Antirrhinum majus, floral dorsoventral symmetry depends on the activity of two TCP-box genes, CYCLOIDEA (CYC) and DICHOTOMA (DICH). To examine whether the same molecular mechanism of floral asymmetry operates in the distantly related Rosid clade of eudicots, in which asymmetric flowers are thought to have evolved independently, we investigated the function of a CYC homologue LjCYC2 in a papilionoid legume, Lotus japonicus. We showed a role for LjCYC2 in establishing dorsal identity by altering its expression in transgenic plants and analyzing its mutant allele squared standard 1 (squ1). Furthermore, we identified a lateralizing factor, Keeled wings in Lotus 1 (Kew1), which plays a key role in the control of lateral petal identity, and found LjCYC2 interacted with Kew1, resulting in a double mutant that bore all petals with ventralized identity to some extents. Thus, we demonstrate that CYC homologues have been independently recruited as determinants of petal identities along the dorsoventral axis in two distant lineages of flowering plants, suggesting a common molecular origin for the mechanisms controlling floral zygomorphy.

New insights into the complex and coordinated transcriptional regulation networks underlying rice seed development through cDNA chip-based analysis.

Transcription factors (TFs) are major, crucial factors for developmental control. To elucidate the effects of TFs on rice seed development, we generated a cDNA chip containing 325 rice cDNA clones, which are from flowering stage and encode known or putative TFs belonging to 12 different families, and used this chip for expression profiling at 8 continuous seed developmental stages. The results showed that in comparison to their expression in mature leaves, a total of 135 TF genes were preferentially transcribed in seeds. Cluster analysis based on the temporal expression patterns grouped them into 12 types, each of which contained members of various families showing common unique expression patterns. The results provide insights into possible key roles for members of several TF families during seed development. In addition, the expression patterns of these genes were examined in vegetative tissues including roots, seedlings and stems, as well as in 2-week-old seedlings following the application of plant hormones or abiotic stresses. The results showed that many of the seed-preferential TFs were also involved in hormone and/or abiotic stress effects, suggesting the potential existence of uncharacterized transcriptional networks, or cross talk between hormone and abiotic stress signaling and seed development. Furthermore, analysis on the cis-elements locating in promoter region of seed preferential TF genes suggested that Dof proteins play essential roles in hierarchical regulation of gene expressions during rice seed development, which, taken together, provided informative clues for elucidation of the molecular mechanisms of transcriptional regulation and signaling networks in the complex developmental processes of rice seeds.